Project Summary/Abstract The lens of the eye is a complex organ that, with the cornea, focuses an image on the retina. Lens cells have glucose transporters, chloride channels, sodium and potassium channels, gap junctions, and many other standard transport proteins in common with the cells of other organs. Aquaporin zero (AQP0) however is a unique water channel protein found only in the fiber cells of the lens. This proposal examines the roles of AQP0, in particular ionic regulation of its water permeability (Pf) by Ca2+ (mediated by calmodulin, CaM) and pH. We use a combination of biophysics (with molecular biology to design and construct informative mutants of AQP0 and measurement of Pf), atomistic molecular dynamic simulation (with simulations designed to investigate the influence of CaM and pH on Pf), and the effects of adding, altering or deleting AQP0 in the lens of living zebrafish. These three aims mesh together as the ?Triangle of Investigation? with each aim informing and supporting the other two. In the zebrafish, there are two AQP0s, one (Aqp0a) a water channel and one not a water channel (Aqp0b) but still essential for lens clarity and development. An aquaporin from a distantly related teleost fish (MIPFun) can supply the functions of both Aqp0a and AQp0b and this allows us to dissect out both the Pf function of AQP0 and its other possible functions such as adhesion, interaction with structural proteins and so forth. Our work is leading to a better understanding of lens physiology and the molecular mechanisms of water channel gating, and offers the hope of delaying or eliminating the formation of cataracts.